Automated evaluation of autoantibodies on human epithelial-2 cells as an approach to standardize cell-based immunofluorescence tests

INTRODUCTION: Analysis of autoantibodies (AAB) by indirect immunofluorescence (IIF) is a basic tool for the serological diagnosis of systemic rheumatic disorders. Automation of autoantibody IIF reading including pattern recognition may improve intra- and inter-laboratory variability and meet the demand for cost-effective assessment of large numbers of samples. Comparing automated and visual interpretation, the usefulness for routine laboratory diagnostics was investigated. METHODS: Autoantibody detection by IIF on human epithelial-2 (HEp-2) cells was conducted in a total of 1222 consecutive sera of patients with suspected systemic rheumatic diseases from a university routine laboratory (n = 924) and a private referral laboratory (n = 298). IIF results from routine diagnostics were compared with a novel automated interpretation system. RESULTS: Both diagnostic procedures showed a very good agreement in detecting AAB (kappa = 0.828) and differentiating respective immunofluorescence patterns. Only 98 (8.0%) of 1222 sera demonstrated discrepant results in the differentiation of positive from negative samples. The contingency coefficients of chi-square statistics were 0.646 for the university laboratory cohort with an agreement of 93.0% and 0.695 for the private laboratory cohort with an agreement of 90.6%, P < 0.0001, respectively. Comparing immunofluorescence patterns, 111 (15.3%) sera yielded differing results. CONCLUSIONS: Automated assessment of AAB by IIF on HEp-2 cells using an automated interpretation system is a reliable and robust method for positive/negative differentiation. Employing novel mathematical algorithms, automated interpretation provides reproducible detection of specific immunofluorescence patterns on HEp-2 cells. Automated interpretation can reduce drawbacks of IIF for AAB detection in routine diagnostics providing more reliable data for clinicians

Proteinkinase C-dependent Regulation of Vesicleformation at the Trans-Golgi- Network

Titelblatt und Inhalt 1 Einleitung 1 2 Aufgabenstellung 25 3 Material 26 4 Methoden 33 5 Ergebnisse und Diskussion 50 6 Zusammenfassung 98 7 Summary 99 8 Tabellen 100 9 Literatur 106 </FONTPKC stimuliert am trans-Golgi-Netzwerk (TGN) die Bildung von konstitutiven Transportvesikeln [165]. Zur Aufklärung des Wirkungsmechanismus wurden zwei Wege beschritten: Zunächst wurden Bindungsproteine der PKC mit Hilfe der "overlay"-Technik analysiert. Durch hochauflösende 2D-Elektrophorese und Mikrosequenzierung wurde Golgi-assoziertes b -Aktin als Hauptbindungsprotein von aktivierter PKCa am Golgiapparat (GA) nachgewiesen. In weiteren Experimenten wurden Golgiproteine an isolierten Golgizisternen oder in permeabilisierten Zellen phosphoryliert. Die erhaltenen Phosphoproteine wurden von nicht phosphorylierten Proteinen durch zweidimensionale Elektrophorese nach Hartinger et al. [259] und Görg et al. [260,303] getrennt. Proteine, deren Phosphorylierung durch Calphostin C, Ro 31-8220 und Gö 6976 gehemmt werden konnte, wurden sequenziert. Von den identifizierten in-vitro-PKC- Substraten MARCKS, MacMARCKS, der regulatorischen leichten Kette des Myosinkomplexes 2A, Cytokeratin 8 und Cytokeratin 18 konnte auch deren in- situ-Phosphorylierung in permeabilisierten Zellen nachgewiesen. Damit ist eine biologische Relevanz der Phosphorylierung dieser Proteine wahrscheinlich. Zusätzlich konnten die bekannten Golgi-assoziierten Proteine wie Rab6, Rab8 und Synaptobrevin 2 identifiziert und andere wie Annexin IV, Profilin I, Rab7, GRP 78 und Endobrevin erstmals am GA nachgewiesen werden. Der Einfluß von Annexin IV und Profilin I auf die in-vitro-Vesikelbiogenese wurde überprüft: Während ein Annexin IV-spezifischer Antikörper die Verpackung von HSPG im zellfreien System nicht beeinflußte [371], konnte durch Zusatz von spezifischem Profilin I-Antikörper die Vesikelbildung gehemmt werden [368]. Profilin I wurde daneben auch biochemisch in post-Golgi-Vesikelfraktionen nachgewiesen, was die oben gemachte Aussage unterstützt. Die Identifizierung der hauptsächlichen PKC-Substrate MARCKS, MacMARCKS und der regulatorischen leichten Kette des Myosinkomplexes 2A deutet auf einen neuen Signaltransduktionsweg hin, durch den PKC die Vesikelbildung am TGN steuern könnte.PKC is known to stimulate the formation of constitutive transport vesicles at the trans Golgi network [165]. In an attempt to understand the mechanism behind that process, two possible experimental approaches were followed: In the first approach PKC-binding proteins at the Golgi apparatus (GA) were analyzed according to a PKC-overlay assay technique. b -Actin was identified as a major binding protein of activated PKCa by high resolution 2D- electrophoresis and microsequencing techniques. In the second experimental setup isolated Golgi cisternae or permeabilized HepG2-cells were used to phosphorylate and separate Golgi proteins by high resolution 2D- electrophoresis according to Hartinger et al. [259] and Görg et al. [303] in the presence of PKCa [260]. Proteins not phosphorylated in the presence of Calphostin C, Ro 31-8220 and/or Gö 6976, were sequenced by mass spectroscopy (MALDI-MS and Q-TOF). For the identified in vitro PKC-substrates MARCKS, MacMARCKS, Myosin RLC, Cytokeratin 8 and Cytokeratin 18, it was possible to demonstrate their phosphorylation in permeabilized HepG2 cells too. Therefore, a biological relevance of this phosphorylation can be assumed. Other known Golgi associated proteins like Rab-6, Rab-8 and VAMP-2 were also identified or were shown to be Golgi associated for the first time as in the case of Annexin-IV, Profilin-I, Rab-7, GRP-78 and Endobrevin. Additionally Annexin IV and Profilin I were tested for their effect on in-vitro-vesicle biogenesis: Even though an Annexin IV-specific antibody did not influence the budding efficacy of HSPG in the cell free system at all [371], the addition of a Profilin I-specific antibody did actually reduce the budding efficiency [368]. Supporting the above result, Profilin I was also identified in post-Golgi- vesicle fractions biochemically. Generally the identification of the mayor PKC substrates MARCKS, MacMARCKS and Myosin RLC is pointing towards a new signal transduction pathway, by which PKC might regulate the vesicle biogenesis at the TG